The present invention describes in vitro and in vivo production of platelets and/or proplatelets from megakaryocytes. The present invention is also directed to compositions comprising megakaryocytes, nitric oxide donors (i.e. compounds that donate, transfer or release nitric oxide, elevate endogenous levels of endothelium-derived relaxing factor, stimulate endogenous synthesis of nitric oxide or are substrates for nitric oxide synthase), and, optionally, at least one thrombopoiesis stimulating factor. The thrombopoiesis stimulating factor is preferably thrombopoietin. The nitric oxide donor is preferable S-nitrosoglutathione. The present invention also describes compositions comprising at least one nitric oxide donor and at least one thrombopoiesis stimulating factor. The present invention also provides methods for treating and/or preventing blood platelet disorders, and for producing platelets and/or proplatelets in vitro and in vivo. The compounds and/or compositions of the present invention can be provided in the form of a pharmaceutical kit.
Platelets are circulating cell derived fragments that are required for the maintenance of hemostasis. These small, anucleate fragments represent the first line of defense against hemorrhage following vascular injury, and are crucial for blood coagulation. Platelets are the terminal differentiation product of megakaryocytes, which in turn originate from pluripotent stem cells. The process of platelet production from megakaryocytes, which is complex and incompletely understood, is called thrombopoiesis. Several cytokines have been reported to stimulate the growth and maturation of megakaryocytes. The interaction between the cytokines and growth factors, their kinetic choreography, and the specific molecular steps that commit the megakaryocytes and their precursors to the process of maturation and platelet production have only begun to be rigorously investigated. Megakaryocytes mature by a process of endomitosis and cytoplasmic maturation. Most research to date has focused on the maturation step of megakaryocyte growth rather than on the terminal process of platelet production.
Morphological studies of marrow megakaryocytes suggest that platelets form as a result of cytoplasmic fragmentation. With the completion of endomitosis, megakaryocyte cytoplasm expands and, in the process, develops demarcation membranes and granules. Platelets form as the fully mature megakaryocyte develops cytoplasmic extensions, or pseudopodial protrusions, that extend in proximity to sinusoidal endothelial cells (Tavassoli and Aoki, Blood Cells, 15:3-14, (1989)). Platelets bud from the ends of these protusions and thereafter enter the circulation. The megakaryocyte""s ability to produce platelet buds is ultimately exhausted, and it undergoes terminal apoptosis.
The in vitro counterpart to thrombopoiesis is believed to be the development of the xe2x80x9cproplateletxe2x80x9d process that has been observed in the terminal phases of megakaryocyte tissue cultures (Choi et al, Blood, 85:402-413 (1995)). Some data suggests that proplatelets can produce platelet-like particles (Choi et al, Blood, 402-413 (1995); Zeigler et al, Blood, 84:4045-4052 (1994)). Proplatelets insinuating between bone marrow sinusoidal cells can enter the circulation (Tavassoli et al, Blood Cells, 15:3-14 (1989)). Circulatory shear forces within the marrow or possibly in the pulmonary circulation could result in the fragmentation of these proplatelets, thereby producing platelets in circulation (Burstein et al, Magakaryopoiesis and Platelet Formation, McGraw-Hill, New York, (1995); Trowbridge et al, Thromb Res., 28:461-475 (1982)).
A number of diseases or conditions result from inappropriate levels or inadequate functioning of blood platelets. Platelet disorders are clinically treated by administering thrombopoietin or by whole blood or platelet transfusions. Platelets for such procedures are obtained by plateletphoresis from normal donors; however, blood and platelet supplies can be limited. In addition, platelets have a relatively short shelf-life of about 5 days. Transfusions are also costly and can transmit infections and expose patients to viruses such as the human immunodeficiency virus (HI) or various hepatitis viruses. Furthermore, patients are often refractory to subsequent transfusions. Thrombopoietin treatment has a lag period before the level of platelets are affected and often results in the failure to stimulate platelet production in many patients.
Thus, there remains a need in the art for new and improved methods for in vitro production of platelets for use by patients and for new and improved methods of stimulating or enhancing the production of platelets in vivo, thereby resulting in safer alternatives for treating and/or preventing blood platelet disorders. The present invention is directed to these, as well as other, important ends.
Nitric oxide (NO) has been shown to mediate a number of actions, including the bactericidal and actions of macrophages and blood vessel relaxation of endothelial cells. NO and NO donors have also been implicated as mediators for a number of processes including vasodilation, neurotransmission, immunity, and vascular and nonvascular smooth muscle relaxation. In the process of arriving at the present invention, it was hypothesized that when the megakaryocytes are committed to platelet production, an endothelial product is released from marrow sinusoidal endothelial cells as they make contact with megakaryocyte protrusions. This endothelium product should promote apoptosis, since platelet formation morphologically resembles programmed cell death (Radley and Hailer, Br. J. Haematol., 53:227-287 (1983)). One possible endothelial product that promotes apoptosis is nitric oxide.
One aspect of the present invention provides compositions comprising at least one megakaryocyte and at least one compound that donates, transfers or releases nitrogen monoxide as a charged species, i.e., nitrosonium (NO+) or nitroxyl (NOxe2x88x92), or as the neutral species, nitric oxide (NOxc2x7), and/or stimulates endogenous production of nitric oxide or endothelium-derived relaxing factor (EDRF) in vivo and/or is a substrate for nitric oxide synthase (i.e., nitric oxide donor). Preferably, the nitric oxide donor is S-nitroso-glutathione. The compositions can comprise a pharmaceutically acceptable carrier. These compositions, which are useful for the in vitro production of platelets and/or proplatelets, can be produced by treating megakaryocytes in culture with an effective amount of at least one nitric oxide donor. These compositions potentiate apoptosis of the cells and increase the number of platelets and/or proplatelets produced.
Another aspect of the present invention provides compositions comprising at least one megakaryocyte, at least one thrombopoiesis stimulating factor, and at least one compound that donates, transfers or releases nitrogen monoxide as a charged species, i.e., nitrosonium (NO+) or nitroxyl (NOxe2x88x92), or as the neutral species, nitric oxide (NOxc2x7), and/or stimulates endogenous production of nitric oxide or EDRF in vivo and/or is a substrate for nitric oxide synthase. The thrombopoiesis stimulating factor is preferably thrombopoietin (TPO), and the nitric oxide donor is preferably S-nitroso-glutathione. The compositions can comprise a pharmaceutically acceptable carrier. These compositions, which are useful for the in vitro production of platelets and/or proplatelets, can be produced by treating megakaryocytes in culture with an effective amount of at least one thrombopoiesis stimulating factor and at least one nitric oxide donor. Preferably, the compositions are produced by treating megakaryocytes in culture with an effective amount of at least one thrombopoiesis stimulating factor followed by treatment of the cells with at least one nitric oxide donor. These compositions increase the production of platelets and/or proplatelets in culture.
Another aspect of the present invention provides methods for stimulating the in vivo production of a patient""s own platelets and/or proplatelets by administering to a patient a therapeutically effective amount of at least one thrombopoiesis stimulating factor and at least one compound that donates, transfers or releases nitrogen monoxide as a charged species, i.e., nitrosonium (NO+) or nitroxyl (NOxe2x88x92), or as the neutral species, nitric oxide (NOxc2x7), and/or stimulates endogenous production of nitric oxide or EDRF in vivo and/or is a substrate for nitric oxide synthase, thereby augmenting the production of the patient""s own platelets and/or proplatelets. The thrombopoiesis stimulating factor and nitric oxide donor can be administered separately or as components of the same composition in one or more pharmaceutically acceptable carriers.
Another aspect of the present invention provides methods for treating and/or preventing blood platelet disorders in a patient by administering a therapeutically effective amount of at least one compound that donates, transfers or releases nitrogen monoxide as a charged species, i.e., nitrosonium (NO+) or nitroxyl (NOxe2x88x92), or as the neutral species, nitric oxide (NOxc2x7), and/or stimulates endogenous production of nitric oxide or EDRF in vivo and/or is a substrate for nitric oxide synthase, and, optionally, at least one thrombopoiesis stimulating factor. The thrombopoiesis stimulating factor and nitric oxide donor can be administered separately or as components of the same composition in one or more pharmaceutically acceptable carriers. The nitric oxide donor and, optionally, at least one thrombopoiesis stimulating factor can also be administered in combination with other medications for the treatment of blood platelet disorders. These methods of treating and/or preventing platelet disorders are preferable to current transfusion therapies which are vehicles for infections, such as AIDS and hepatitis.
Another aspect of the present invention provides methods for treating and/or preventing blood platelet disorders in a patient by administering platelets and/or proplatelets produced by culturing megakaryocytes in vitro. The megakaryocytes in culture are treated with an effective amount of at least one compound that donates, transfers or releases nitrogen monoxide as a charged species, i.e., nitrosonium (NO+) or nitroxyl (NOxe2x88x92), or as the neutral species, nitric oxide (NOxc2x7), and/or stimulates endogenous production of nitric oxide or EDRF in vivo and/or is a substrate for nitric oxide synthase, and, optionally, at least one thrombopoiesis stimulating factor. The thrombopoiesis stimulating factor and nitric oxide donor can be administered separately or as components of the same composition in one or more pharmaceutically acceptable carriers. The megakaryocytes in culture can be derived from a variety of sources, such as cell lines, stem cells, tissues, bone marrow or a patient""s own blood megakaryocytic precursors. These methods of treating platelet disorders are preferable to current transfusion therapies which are vehicles for infections, such as AIDS and hepatitis.
Yet another aspect of the present invention provides methods for reducing high platelet counts in a patient by administering an effective amount of at least one compound that inhibits the patient""s production of nitric oxide.
These and other aspects of the present invention are described in detail below.